Dual-targeting liposome modified by glutamic hexapeptide and folic acid for bone metastatic breast cancer

Article abstract of DOI:10.1016/j.chemphyslip.2020.104882

Bone is the most common organ affected by metastatic breast cancer. Targeting delivery of drugs to bone may not only enhance the treatment efficacy, but also reduce the quantity of drug administered. In order to increase the distribution of paclitaxel (PTX) in bone, herein, a novel bone metastasis-targeted glutamic hexapeptide-folic acid (Glu₆-FA) derivative was designed and synthesized as liposome ligand to deliver PTX to bone metastasis effectively. The liposomes were prepared by thin film hydration method and its particle size, zeta potential, encapsulation efficiency, release profile, stability, hemolysis were also characterized. What's more, the anti-tumor effects of PTX-Glu₆-FA-Lip were confirmed by the detection of cell cycle, migration, and further measurement of microtubule stabilization. In addition, the PTX-Glu₆-FA-Lip showed superior targeting ability in vitro and in vivo evaluation as compared to naked PTX, non-coated, singly-modified and co-modified by physical blending liposomes. All the results suggested that Glu₆-FA-modified liposome showed excellent targeting activity to metastatic bone cancer. These findings suggested that Glu₆-FA-Lip was a promising bone metastasis-targeting carrier for the delivery of PTX. This study may therefore be conducive to the field of bone-targeting drugs delivery.

Full text of DOI:10.1016/j.chemphyslip.2020.104882

ChemistryandPhysicsofLipids228(2020)104882
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Dual-targeting liposome modified by glutamic hexapeptide and folic acid for
bone metastatic breast cancer
Yang Yanga, Ze Zhaob, Changwei Xieb, Yi Zhaoa,*
a Department of Translational Medicine Center, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450052, China
b Department of Orthopedics, the First Affiliated Hospital of Henan Polytechnic University (the Second People's Hospital of Jiaozuo City), Jiaozuo 454001, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Bone is the most common organ affected by metastatic breast cancer. Targeting delivery of drugs to bone may
not only enhance the treatment efficacy, but also reduce the quantity of drug administered. In order to increase
the distribution of paclitaxel (PTX) in bone, herein, a novel bone metastasis-targeted glutamic hexapeptide-folic
acid (Glu6-FA) derivative was designed and synthesized as liposome ligand to deliver PTX to bone metastasis
effectively. The liposomes were prepared by thin film hydration method and its particle size, zeta potential,
encapsulation efficiency, release profile, stability, hemolysis were also characterized. What’s more, the anti-
tumor effects of PTX-Glu6-FA-Lip were confirmed by the detection of cell cycle, migration, and further mea-
surement of microtubule stabilization. In addition, the PTX-Glu6-FA-Lip showed superior targeting ability in vitro
and in vivo evaluation as compared to naked PTX, non-coated, singly-modified and co-modified by physical
blending liposomes. All the results suggested that Glu6-FA-modified liposome showed excellent targeting activity
to metastatic bone cancer. These findings suggested that Glu6-FA-Lip was a promising bone metastasis-targeting
carrier for the delivery of PTX. This study may therefore be conducive to the field of bone-targeting drugs
delivery.
Bone metastasis
Bone-targeting
Drug delivery
Glutamic hexapeptide
Folic acid
1. Introduction
metastasis, bone-targeting drug delivery is a promising solution to in-
crease therapeutic efficiency and avoid the severe side effects (Zhu
Bone diseases, such as bone metastasis, are notoriously difficult
diseases to treat due to the comparatively low blood flows in bone
tissue (Ellmann et al., 2019). Bone metastasis is a frequent complication
in advanced stage of many cancers, especially breast, prostate, and lung
cancers (Lee et al., 2018; Rong et al., 2018; Sun et al., 2019). It could
give rise to bone pain, hypercalcemia, pathological fracture and bone
malformation because of the increased activity of osteoclast cells,
which leads to the decrease of life quality (Ferrer Albiach et al., 2019;
Yeo et al., 2015). Current treatments for metastatic bone cancers mainly
include surgery, radiotherapy and chemotherapy, or the combination of
them (Gnant et al., 2012). Although the chemotherapy has played a
vital role in increasing the survival rate and improving the life quality
of patients, its low targeting efficiency makes the high and frequent
administration essential, which may lead to severe side effects (Li et al.,
2017; Wang et al., 2014). Therefore, there is a huge amount of demand
of specific delivery systems of chemotherapeutic agents, such as pacli-
taxel (PTX), to the site of bone metastasis lesion are of great interests for
the treatment of metastatic bone cancer.
et al., 2018). As we all know, bone is consist of some unique materials,
and the inorganic compound hydroxyapatite (HAP) is the major in-
gredient of bone, which is a potential target for selective drug delivery
to bone. In our previous study, we have found that glutamic oligo-
peptide, especially glutamic hexapeptide, had an excellent bone-tar-
geting ability (Zhao et al., 2015). According to Sarig’s research, the
mechanisms for the bone-affinity of glutamic oligopeptide is that the
multi-carboxy groups of peptides provide ionic interaction between
negative charges and calcium ions in the mineral component (HAP) of
bone, and this kind of affinity only depends on the number of exposed
amino acid residues (Sarig, 2004).
What’s more, it is well known that folic acid (FA) is one of the most
frequently-used neoplastic targeting ligands in drug delivery system
(Papaioannou et al., 2018; Park et al., 2019). Folic acid is a targeting
ligand used for the delivery of anti-cancer drugs, and the folate re-
ceptors (FRs) are over expressed in many tumor sites including breast,
ovarian, lung, and colon cancers (Diaz-Diestra et al., 2018; Handali
et al., 2018; He et al., 2015; Nie et al., 2018). As a results, a lot of
interesting researches had been done on the nanocarriers modified by
To overcome the limitations of chemotherapy for treating bone
⁎ Corresponding author.
E-mail address: zhaoyi0910@163.com (Y. Zhao).
https://doi.org/10.1016/j.chemphyslip.2020.104882
Received 24 September 2019; Received in revised form 5 January 2020; Accepted 29 January 2020
Availableonline01February2020
0009-3084/©2020ElsevierB.V.Allrightsreserved.

Y. Yang, et al.
ChemistryandPhysicsofLipids228(2020)104882
folic acid to deliver drug to target the tumors (Papaioannou et al., 2018;
Park et al., 2019). In addition, the folate receptor is also highly ex-
pressed in bone metastatic cells and osteoclasts (Nagai et al., 2012), so
that the folate receptor is also an attractive target for bone.
2. Materials and methods
2.1. Chemistry
As a drug carrier, liposome has many advantages such as non-
toxicity, biocompatibility and biodegradability, which is widely applied
in drug delivery system (Kraft et al., 2014). Cholesterol is a common
additive in the preparation of liposomes because of its good bio-
compatibility and strong hydrophobicity (Axmann et al., 2019). It can
regulate the fluidity of the bilayer of liposome phospholipids, reduce
the permeability of the membrane, reduce the leakage of drugs, protect
the oxidation of phospholipids, and enhance the flexibility of the
membrane so that to increase the stability of the liposome. Over the
years, we has done many researches on bone targeting and cancer
targeting by active process (Zhao et al., 2015). And we are still ex-
ploring how to further improve the carrier's bone metastasis targeting
ability to deliver more drugs to bone. As parts of our continuing efforts,
we focused on utilizing glutamic hexapeptide and folic acid to selec-
tively deliver drug to bone metastasis. Herein, we developed a PTX-
loaded liposome co-modified with glutamic hexapeptide and folic acid.
Such delivery system had a potential bone-targeted affinity through the
Glu6 and FA exposed on the surface of the liposome, and facilitated
bone metastasis cellular uptake by the modification of FA (Fig. 1). The
Glu6-FA modified, non-coated, singly-modified and co-modified by
physical blending liposomes (Glu6+FA) were prepared by the lipid film
hydration-ultrasound method. Using post-insertion technique, the li-
pophilic steroidal portion was embedded into the liposome membrane,
while the hydrophilic Glu6-FA residues were exposed outside the
membrane. Through the wound closure and flow cytometry (FCM) as-
says, we found the obvious impaired migration capacity of breast
cancer cells and the cell cycle arrest caused by the treatment of PTX-
Glu6-FA-Lip. Interestingly, the more obvious effects on microtubule
stabilization was also found through microtubule depolymerization
experiments, which further confirmed the impairment of cell migration
and cell cycle. Additionally, the bone targeting in vitro and in vivo was
also investigated.
2.1.1. Synthesis of compound 3–11
The synthesis of compound 3–11 was reported in our previous work
(Fu et al., 2019; Zhao et al., 2014, 2015; Zhao et al., 2018; Zhao et al.,
2020).
2.1.2. Synthesis of compound 12
To a solution of compound 5 (0.50 g, 0.33 mmol) in CH2Cl2 (20 mL)
were added dicyclohexylcarbodiimide (DCC, 67 mg, 0.33 mmol) and
cat. dimethylaminopyridine (DMAP), then the mixture was stirred at
−5 °C for 30 min. Compound 11 (0.22 g, 0.33 mmol) in CH2Cl2 (5 mL)
was added to the above reaction mixture slowly. After stirring for 20 h
at room temperature, the reaction was terminated and then filtered.
The filtrate was concentrated, and the residue was purified by flash
chromatography to give 12 (0.41 g, 57 %) as white waxy solid. HRMS:
(ESI+) calculated for C122H157N7O28Na [M + Na]+ 2192.1008, found
2192.1005. Elemental Analysis: C, 67.54; H, 7.29; N, 4.52, found C,
67.42; H, 7.37; N, 4.68.
2.1.3. Synthesis of compound 13
First, folic acid (0.30 g, 0.68 mmol), compound 12 (0.74 g, 0.34
mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 53 mg,
0.34 mmol) and N-hydroxysuccinimide (NHS, 47 mg, 0.41 mmol) were
dissolved in 10 mL dimethyl sulfoxide (DMSO), and the mixture was
stirred at room temperature for 24 h. The byproduct, dicyclohexylurea,
was removed by filtration, and the filtrate was precipitated by diethyl
ether to obtain a yellow solid, which was purified by flash chromato-
graphy to give 13 (0.12 g, 31 %) as yellow waxy solid. HRMS: (ESI+)
calculated for
C141H174N14O33Na [M +
Na]+ 2615.2299, found
2615.2294. Elemental Analysis: C, 65.31; H, 6.76; N, 7.56, found C,
65.43; H, 6.85; N, 7.41.
2.1.4. Synthesis of ligand Glu6-FA-Chol
To a solution of compound 13 (65 mg, 0.025 mmol) in CH3OH (5
mL), Pd/C (20 mg, 10 %) was added. Then, the mixture was stirred in
hydrogen atmosphere at 5 °C for 2 h. Pd/C was filtered, and the filtrate
Fig. 1. Schematic illustration of PTX-loaded Glu6-FA-Lip to target metastatic tumor cells.
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Y. Yang, et al.
ChemistryandPhysicsofLipids228(2020)104882
was concentrated to give ligand Glu6-FA-Chol (25 mg, 48 %) as yellow
waxy solid. HRMS: (ESI+) calculated for C92H132N14O33Na [M + Na]+
1983.8979, found 1983.8983. Elemental Analysis: C, 56.32; H, 6.78; N,
9.99, found C, 56.51; H, 6.62; N, 9.84.
mm, 5 μm, SinoChrom) maintaining at 30 °C. The mobile phase con-
sisted of water-methanol (67:33, v/v) with a low flow rate 1.0 mL/min.
The sample volume injected was 20 μL and the detection wavelength
was 227 nm. The EE% and DL% were calculated using the following
equations: EE% = weight of encapsulated PTX / the total weight of
PTX, DL % = weight of encapsulated PTX / the total weight of PTX-
loaded liposome. The particle size and zeta potential of Lip, Glu6-Lip,
FA-Lip, Glu6 + FA-Lip and Glu6-FA-Lip were characterized by Malvern
Zeta sizer Nano ZS90 (Malvern Instruments Ltd., UK).
2.1.5. Synthesis of ligand FA-Chol
To a solution of folic acid (1.00 g, 2.26 mmol) and compound 8
(0.58 g, 1.13 mmol) in DMSO 20 mL was added EDC (0.17 g, 1.13
mmol) NHS (0.16 g, 1.36 mmol). Then, the mixture was stirred at room
temperature for 24 h. The byproduct was removed by filtration. The
filtrate was precipitated by diethyl ether to obtain a yellow solid, which
was purified by flash chromatography to give FA-Chol (0.37 g, 35 %) as
yellow solid. HRMS: (ESI+) calculated for C52H75N7O9Na [M + Na]+
964.5524, found 964.5525. Elemental Analysis: C, 66.29; H, 8.02; N,
10.41, found C, 66.20; H, 8.15; N, 10.32.
2.3. In vitro paclitaxel release study of liposomes
The paclitaxel release analysis from PTX-loaded liposomes was
performed in PBS (pH 7.4) using dialysis method. Briefly, each PTX
liposomal formulation (0.4 mL) and free paclitaxel (dissolved in
ethanol/polyoxyethylated castor oil: v/v = 1/1) were put into a dia-
lysis bag (MWCO = 8 000–14 000 Da) placed in 40 mL release medium
(PBS containing 0.1 % (v/v) Tween 80). It was gently oscillated at 37 °C
and 0.1 mL sample was taken out and replaced with fresh medium at
predetermined time points (0 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h).
Then, the amount of PTX was determined using HPLC method as
mentioned above.
2.1.6. Synthesis of compound 14
To a solution of compound 10 (1.00 g, 1.73 mmol) in CH2Cl2 (25
mL) was added N-methylmorpholine (NMM, 0.29 mL, 2.60 mmol) and
isobutyl chlorocarbonate (IBCF, 0.26 mL, 2.08 mmol), and the reaction
was stirred at −10 °C for 15 min. Then compound 4 (2.22 g, 1.56
mmol) in CH2Cl2 (10 mL) was added slowly. After stirring for another
10 h at r.t., the mixture was washed with 1 mol/L HCl, saturated
NaHCO3 and saturated NaCl. The organic layer was dried over anhy-
drous Na2SO4, and concentrated in vacuo. The residue was purified by
flash column chromatography to afford 14 (1.75 g, 56 %) as white waxy
solid. HRMS: (ESI+) calculated for C114H144N6O24Na [M + Na]+
2005.0163, found 2005.0160. Elemental Analysis: C, 69.07; H, 7.32; N,
4.24, found C, 69.18; H, 7.45; N, 4.39.
2.4. In vitro stability of liposomes in serum
To investigate the stability of PTX liposomal formulations, the tur-
bidity variations in the presence of fetal bovine serum (FBS) and the
appearance In brief, 0.1 mL PTX-loaded liposome was incubated in FBS
(0.1 mL) with moderate shaking (45 rpm) at 37 °C. The transmittance of
each sample was detected at the predetermined time points (0 h, 1 h, 2
h, 4 h, 8 h, 12 h, 24 h and 48 h) using a microplate reader (Thermo
Scientific Varioskan Flash, USA) at 750 nm. What’s more, the stability
of liposomes stored at 4 °C for several days was also investigated by
monitoring their particle size and polydispersity index (PDI) at the
predetermined time points.
2.1.7. Synthesis of ligand Glu6-Chol
To a solution of compound 14 (0.10 g, 0.050 mmol) in CH3OH (5
mL), Pd/C (20 mg, 10 %) was added. Then, the mixture was stirred in
hydrogen atmosphere at 5 °C for 2 h. Pd/C was filtered, and the filtrate
was concentrated to give ligand Glu6-Chol (46 mg, 68 %) as white waxy
solid. HRMS: (ESI+) calculated for C65H102N6O24Na [M + Na]+
1373.6843, found 1373.6840. Elemental Analysis: C, 57.76; H, 7.61; N,
6.22, found C, 57.66; H, 7.52; N, 6.37.
2.5. Hemolysis assays
For hemolysis assays, the mouse red blood cells (MRBCs) were ob-
tained according to our previous report (Fu et al., 2019). Briefly, the
fresh mouse blood was stabilized and centrifuged at 5 × 103 rpm for 5
min to remove the supernatant. Afterwards, the precipitated MRBCs
were washed with PBS, and then diluted with PBS to a concentration of
2 % (w/v). Subsequently, 0.1 mL MRBCs solution was added into dif-
ferent liposomes with 0.4 mL various concentrations of lipids (10−400
nM). MRBCs incubated with PBS and Triton X-100 were utilized as
negative and positive controls, respectively. All the mixtures were in-
cubated at 37 °C for 2 h with gentle shaking, and were centrifuged at 1
× 104 rpm for 10 min. Finally, the absorbance of the supernatant was
measured using a microplate reader (Thermo Scientific Varioskan
Flash) at 540 nm. The hemolytic activity percentages of the different
samples were calculated using the following equation: the percent he-
molysis (%) = (ASample-ANegative)/(APostive-ANegative) × 100 %, where A
is the absorbance of hemoglobin.
2.2. Preparation and characterization of liposomes
PTX-loaded liposomes were prepared through thin film hydration
method as our previous reported (Fu et al., 2019), including a main
lipid composition of SPC, cholesterol, and synthesized ligands. The
component ratio optimized according to our previous report was as
follows: (1) conventional liposomes (Lip), SPC/cholesterol/ (molar
ratio = 62: 33); (2) Ligand Glu6-Chol modified liposomes (Glu6-Lip),
SPC/cholesterol/ligand Glu6-Chol (molar ratio = 62: 33: 3); (3) Ligand
FA-Chol modified liposomes (FA-Lip), SPC/cholesterol/Ligand FA-Chol
(molar ratio = 62: 33: 3); (4) Ligand Glu6-Chol and FA-Chol co-mod-
ified liposomes (Glu6+FA-Lip), SPC/cholesterol/Ligand Glu6-Chol/FA-
Chol (molar ratio = 62: 33: 3: 3); (5) Ligand Glu6-FA-Chol modified
liposomes (Glu6-FA-Lip), SPC/cholesterol/Ligand Glu6-FA-Chol (molar
ratio = 62: 33: 3). In brief, the lipid materials were dissolved in the
chloroform/methanol (v/v = 2:1), and then dried using a rotary eva-
porator at 37 °C, which was further dried in vacuum for 24 h. Subse-
quently, the dried thin film was hydrated with PBS (pH 7.4) at 20 °C for
0.5 h and then sonicated intermittently by a probe sonicator at 80 W for
80 s to form liposomes.
2.6. Hydroxyapatite binding assay
The hydroxyapatite binding assay of these PTX-loaded liposomal
formulations toward bone matrix was conducted according to our
previously reported procedure (Zhao et al., 2015). In brief, the hydro-
xyapatite was dissolved in PBS at the concentration of 20 mg/mL. Then,
0.3 mL PTX-Lip, PTX-Glu6-Lip, PTX-FA-Lip, PTX-Glu6+FA-Lip, PTX-
Glu6-FA-Lip and free PTX (calculated as PTX) were respectively in-
cubated with 0.3 mL HAP suspension and shaken gently at 37 °C. At the
same time, these liposomes were incubated in PBS without HAP used as
negative control. At the predetermined time (2 h or 24 h), the unbound
To prepare PTX-loaded liposomes, appropriate amount of paclitaxel
was added into the lipid organic solution prior to the solvent eva-
poration. Detections about entrapment efficiency (EE%) and drug
loading efficiency (DL %) were conducted using high performance li-
quid chromatography (HPLC). Measurement was performed on a LC-
10A liquid chromatographic system (Shimadzu). The analytical column
was a reverse-phase HPLC column (ODS-C18 column, 4.6 mm × 200
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Y. Yang, et al.
ChemistryandPhysicsofLipids228(2020)104882
PTX was separated from the mixture by centrifugation at 5000 rpm for
3 min. Afterwards, the unbound PTX in the supernatant was determined
quantificationally through HPLC. The binding efficiency was calculated
by HAP binding rate (%) = (C0−C)/C0.
μL propidium iodide (50 μg/mL) for another 30 min. Finally, the cells
were analyzed using a FACS Calibur flow cytometer. The percentage of
cells in G1, S, and G2/M phases was analyzed.
2.10. Wound healing assays
2.7. Cytotoxicity assay
The MDA-MB-231 cells were treated with the PTX-loaded liposomes
(1 μg/mL, calculated as PTX), maintained for 48 h and mechanically
wound was made using a 20-μL pipette tip. Subsequently, the cells were
washed with PBS to remove debris, and the complete culture medium
was added to stimulate wound healing. Photographs were respectively
taken at 0 h and 24 h, and the different PTX formulations of wound
healing was calculated.
MDA-MB-231 cells and MCF10A cells were cultured in Dulbecco’s
Modified Eagles Medium (DMEM) supplemented with 10 % fetal bovine
serum (FBS), 100 U/mL streptomycin and 100 U/mL penicillin at 37 °C
in a humidified incubator containing 5 % CO2 (Thermo Scientific, USA).
The cytotoxicity of PTX-loaded liposomes was measured with MTT
assay. Generally, the cells were seeded into a 96-well plate at a density
of 5 × 103 cells/well, and incubated in a 5 % CO2 humidified en-
vironment at 37 °C for 24 h. PTX-loaded liposomes and blank liposomes
(PTX-unloaded) were diluted to predetermined concentrations with PBS
and added into each well. The final concentrations of PTX were in the
range of 0.016−50 μg/mL, and blank liposomes in the range of lipid
concentration at 0.64−2000 ng/mL. After 24 h incubation, 20 μl of
MTT solution (5.0 mg/mL) was added to each well and the cells were
cultured for another 4 h at 37 °C. Then, the medium were removed and
the dye was dissolved in DMSO (150 μL). The amount of absorption in
each well reflects the conversion of MTT to formazan by metabolically
viable cells, and the cell viability was calculated using the following
equation: ASample / AControl × 100 %, where ASample and AControl, where
2.11. Fluorescence microscopy
The MDA-MB-231 cells grown on glass coverslips treated with the
PTX-loaded liposomes (PTX-Glu6+FA-Lip and PTX-Glu6-FA-Lip) at 1
μg/mL (calculated as PTX), maintained for 48 h. Subsequently, cells
were placed on ice for 0, 15, and 30 min to induce microtubule de-
ploymerization. Then, the cells were fixed with 4 % paraformaldehyde
(PFA) in phosphate-buffered saline (PBS) for 30 min, followed by per-
meabilization with 0.5 % Triton X-100 in PBS for 10 min. Cells were
then blocked with 2 % bovine serine albumin in PBS and incubated
with primary antibodies and then FITC-conjugated secondary anti-
bodies, followed by staining with DAPI. Coverslips were then mounted
and examined with an Axio Observer A1 fluorescence microscope (Carl
Zeiss).
A
Sample and AControl represent the absorbance of sample cells and control
cells read at 490 nm on an automatic microplate spectrophotometer,
respectively.
2.8. Effect of PTX-loaded liposomes on an in vitro bone metastasis model
2.12. Pharmacokinetic study
The in vitro bone metastasis model was established by co-incubating
of MDA-MB-231 cells and hydroxyapatite in three-dimensional collagen
gel to evaluate the effect of PTX-loaded liposomal formulations and free
paclitaxel on bone metastasis from breast cancer. Generally, 4.6 mL
collagen solution (3 mg/mL) was mixed with PBS (0.36 mL, 20×),
NaOH solution (0.64 mL, 4 mg/mL) and 0.36 mL distilled H2O at 4 °C.
Then, the hydroxyapatite particles (10 mg) and MDA-MB-231 cells (5
× 103) were suspended in the above mixed collagen solution (100 μL).
Subsequently, the mixture was transferred to a 96-well plate and cul-
tured at 37 °C for 4 h until complete gelation. Next, the gel was washed
with PBS to expose the hydroxyapatite surface. Then, 100 μL PTX-
loaded liposome formulations (PTX-Lip, PTX-Glu6-Lip, PTX-FA-Lip,
PTX-Glu6+FA-Lip and PTX-Glu6-FA-Lip, equivalent to 20 μg/mL PTX)
were added to the gels and cultured with gently agitation. The PBS
without PTX was used as negative control. After 1 h, the gels was wa-
shed with PBS for thrice and cultured in media (200 μL) for further 24
h. Finally, the cell viability was estimated using MTT assay as described
in section 2.2.7.
Pharmacokinetic study was conducted on healthy Sprague Dawley
(SD) rats and the concentration of PTX was determined by HPLC as
described previously. Briefly, SD rats (180−220 g) were randomly di-
vided into six groups with five rats in each group. Free PTX solution
(dissolved in ethanol/polyoxyethylated castor oil: v/v = 1/1) and PTX-
loaded liposome formulations (PTX-Lip, PTX-Glu6-Lip, PTX-FA-Lip,
PTX-Glu6+FA-Lip and PTX-Glu6-FA-Lip) were intravenous adminis-
tered to each group at an equivalent dose of 10 mg/kg PTX, respec-
tively. Blood samples (0.5 mL) from the orbit were collected at the
predetermined time points (5, 15, 30, 60, 120, 240, 480 and 1440 min)
and centrifuged at 5000 rpm for 5 min at 4 °C to obtain the plasma
samples. Then, the docetaxel internal standard (10 μL, 50 μg/mL) was
added into 100 μL plasma samples, respectively. Extraction was per-
formed with 0.2 mL diethyl ether and the mixture was vortexed for 5
min. After centrifuging at 10 000 rpm for 10 min, the supernatant was
dried at 40 °C under a gentle stream of nitrogen in the pressured gas
blowing concentrator. Then the dried residue was dissolved in me-
thanol (50 μL) and the mixture was further centrifuged at 10 000 rpm
for 10 min. Samples were analyzed by HPLC.
2.9. Cell cycle assays
2.13. Biodistribution and targeting metastatic bone in vivo
Paclitaxel is a well-known anti-cancer agent that inhibits the mi-
crotubule disassembly to arrest cell cycle in the G2/M phase. To go into
the mechanism by which the PTX-Glu6-FA-Lip inhibit cell proliferation,
the percentage of cells in G1, S, and G2/M phases was counted by FACS
analysis. Briefly, the MDA-MB-231 cells were seeded into a 6-well plate
at a density of 5 × 103 cells per well, and then maintained in a 5 % CO2
humidified environment at 37 °C for 24 h. Subsequently, the cells were
treated with different PTX-loaded liposomes at 1 μg/mL final con-
centration of PTX in FBS-free medium. After 0.5 h incubation, the
medium was removed and the cells was washed for approximately 3
times and refreshed with complete medium for another 24 h. Then cells
were collected and washed with PBS. After fixing with 70 % ethyl al-
cohol for 24 h, the cells suspension was centrifuged and washed. Then,
the cells were incubated with RNase A for 30 min and treated with 80
Six-week-old female Balb/c nu mice (18−22 g) bearing MDA-MB-
231 tumors in the tibia were divided into six groups with five mice in
each group. PTX-Lip, PTX-Glu6-Lip, PTX-FA-Lip, PTX-Glu6+FA-Lip,
PTX-Glu6-FA-Lip, and the free paclitaxel solution administered via the
tail vein at an equivalent dose of 10 mg/kg PTX. 0.5 h, 1 h, 2 h, 4 h, 8 h
after administration, the mice were killed by perfusion. The heart, liver,
spleen, lung, kidney, and the lower tibia bone were removed, rinsed
with saline, blotted dry, and weighed. Then, to the 100 μL tissue
homogenate with twice amount of saline was added 10 μL docetaxel (50
μg/mL) as the internal standard, and the mixture was extracted with 0.2
mL diethyl ether. After vortexing for 5 min, the mixture was centrifuged
at 10 000 rpm for 10 min, and the supernatant was dried at 40 °C under
a gentle stream of nitrogen in the pressured gas blowing concentrator.
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Y. Yang, et al.
ChemistryandPhysicsofLipids228(2020)104882
Scheme 1. Synthesis of liposome ligand Glu6-FA-Chol, FA-Chol and Glu6-Chol. Reagents and conditions: (a) i) IBCF, NMM, THF, -15 °C - r.t., 20 h; ii) TFA, CH2Cl2,
r.t., 1 h. (b) Repeat steps as Step (a) for four times. (c) succinic anhydride, CH2Cl2, reflux, 2 h. (d) TsCl, pyridine, 50 °C, 5 h. (e) Triethylene glycol, dioxane, reflux, 6
h. (f) t-butyl bromoacetate, n-Bu4N+HSO4, 50 % NaOH, toluene, r.t., 16 h. (g) TsOH, tolunene, reflux, 8 h. (h) diethanol amine, IBCF, NMM, CH2Cl2, −10 °C - r.t., 5
h. (i) 5, DCC, DMAP, CH2Cl2, −5 °C - r.t., 20 h. (j) folic acid, EDC, NHS, DMSO, r.t., 24 h. (k) H2, Pd/C, CH3OH, 5 °C, 2 h. (l) folic acid, EDC, NHS, DMSO, r.t., 24 h.
(m) 4, IBCF, NMM, CH2Cl2, -15 °C -r.t., 10 h. (n) H2, Pd/C, CH3OH, 5 °C, 2 h.
Then the dried residue was dissolved in methanol (100 μL) and the
mixture was further centrifuged at 10 000 rpm for 10 min. Samples
were analyzed by HPLC.
previous work (Zhao et al., 2015). Cholesterol 6 underwent five steps to
generate compound 11 (Fu et al., 2019). Subsequently, condensation of
compound 11 and Glu6 derivative 5 in the presence of DCC and DMAP
gave 12, which was then coupled with FA to generate 13. Finally, de-
benzylation of the benzyl groups with 10 % Pd/C reached the desired
ligand Glu6-FA-Chol.
2.14. Statistical analysis
What’s more, compound 8 was conjugated with folic acid in the
presence of EDC and NHS to give ligand FA-Chol. Compound 10 was
conjugated with hexapeptide 4 in the presence of IBCF and NMM to
generate compound 14, which was underwent a deprotection reaction
to get the target ligand Glu6-Chol.
All the data were presented as mean standard deviation (SD). The
statistical analyses were conducted using GraphPad Prism 6.0 (San
Diego, CA, USA). Statistical comparisons were performed by analysis of
variance (ANOVA) for multiple groups followed by Student’s t-test. The
significance was defined as follows: *P < 0.05, **P < 0.01,
***P < 0.001.
3.2. Characterization of liposomes
3. Results and discussion
PTX-loaded liposomes were prepared by the thin-film hydration
method. One of the requirments for liposome to target bone is that they
should have proper sizes and uniform distribution. As shown in Table 1,
the dynamic light scattering results suggested that all the liposomes had
the mean diameters about 100−120 nm and low polydispersity index
(PDI close to 0.2). All the Glu6-modified liposomes had the most ne-
gative zeta-potential, while the PTX-Lip showed a slightly negative
3.1. Chemistry
The synthetic pathway of liposome ligand Glu6-FA-Chol was out-
lined in Scheme 1. Briefly, bone-targeting peptide 5 with carboxyl
groups protected by benzyl groups was synthesized by a conventional
liquid-phase peptide synthetic method which was described in our
5

Y. Yang, et al.
ChemistryandPhysicsofLipids228(2020)104882
Table 1
The characterization of different PTX-loaded liposomes (n = 3).
Liposomes
PTX-Lip
PTX-FA-Lip
PTX- Glu6-Lip
PTX-Glu6+FA -Lip
PTX-Glu6-FA -Lip
Size(nm)
PDI
EE (%)
103.5 4.2
105.6 1.9
119.3 3.9
117.4 4.1
114.2 2.9
0.185 0.019
91.85 2.78
3.43 0.23
0.189 0.010
90.52 5.31
3.37 0.17
0.193 0.009
89.21 5.04
3.24 0.25
0.199 0.017
88.67 5.84
3.21 0.18
0.186 0.025
90.17 3.27
3.30 0.31
DL (%)
Zeta potential (mV)
−2.39 0.22
−5.98 0.84
−15.69 2.34
−16.82 3.97
−17.19 2.59
potential. It maybe attribute to the modification with glutamic acid on
the liposome’s surface, which could increase the net negative charge
due to the high density negative carboxyl of glutamic acid and decrease
the absorption of the reticuloendothelial system and immune response
(Murugan et al., 2015). The entrapment efficiency were greater than 86
%, and drug loading efficiency were above 3 %, respectively. All of
results contributed to achieve the passive targeting through the en-
hanced permeability and retention (EPR) effect (Zhang et al., 2009).
In vitro release studies were used to evaluate entrapment, membrane
flexibility and integrity, and affinity of the drug to the carrier systems.
In this study, the PTX release behavior from liposomes was evaluated
using the dialysis method compared with that of the PTX solution (as
control). As shown in Fig. 2A, it was revealed that the PTX solution had
a fast release characteristic as more 80 % of PTX was released within 12
h. While, the result also showed that PTX-loaded liposomes achieved
sustained release behaviors. The PTX-loaded liposome formulations
released less than 60 % of the loaded PTX within 12 h, and the release
exhibited a slow release rate afterwards. What’s more, it was worth
noting that there was no significant difference between PTX-Lip, PTX-
Glu6-Lip, PTX-FA-Lip, PTX-Glu6+FA-Lip and PTX-Glu6-FA-Lip on the
release property. And none of the PTX-loaded liposomes displayed burst
initial release patterns. All the results indicated that the modification on
the surface of liposome didn't change the release profile of PTX.
Liposomal particle stability against physiological condition is pre-
requisite for the further application in vivo. The turbidity variations of
different liposomes were measured to investigate the stability of lipo-
somes in serum. As shown in Fig. 2B, the transmittances of all the li-
posomes were over 90 % and these liposomes didn’t show obvious
change within 48 h cultured with FBS, which suggested that no ag-
gregation or sediment was appeared. On the other hand, the results of
physical stability was shown in Fig. 2C and D. The average size was
almost unchanged at 4 °C for three days (Fig. 1C), and PDI increased
slightly (Fig. 1D). All the results implied that the liposomes were suf-
ficient serum stability, which is important for obtaining a longer half-
life of blood in vivo.
The hemocompatibility of the liposomes drug delivery system was
evaluated using the erythrocyte hemolysis test. As expected, hemolysis
assay of PTX-loaded liposomes demonstrated that the five types of li-
posomes did not show any significant increase in the hemoglobin re-
lease with up to 400 nM of phospholipids (Fig. 2E) and concentration-
dependent increase in hemolysis (< 5 % hemolysis rate was regarded as
non-toxic) (Li et al., 2018), which suggested that they had a good
Fig. 2. (A) The PTX release profiles of free PTX, PTX-Lip, PTX-Glu6-Lip, PTX-FA-Lip, PTX-Glu6+FA-Lip and PTX-Glu6-FA-Lip in PBS (pH 7.4) containing 0.1 % Tween
80 over 48 h. (B) The variations of transmittance of different modified liposomes in 50 % FBS. (C) Size at 4 °C for 72 h. (D) PDI at 4 °C for 72 h. (E) Hemolysis
percentage of different liposomes. (F) HAP binding ratio of different types of PTX-loaded liposomes and PTX after incubation with HAP for 2 h or 24 h. *P < 0.05,
**P < 0.01, **P < 0.001. (n = 3, mean SD).
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Y. Yang, et al.
ChemistryandPhysicsofLipids228(2020)104882
Fig. 3. (A) Cytotoxicity study of blank liposomes against MDA-MB-231 cells. (B) Cytotoxicity study of PTX-loaded liposomes and free PTX against MDA-MB-231 cells.
(C) Cytotoxicity study of PTX-loaded liposomes and free PTX against MCF10A cells. (D) Anti-tumor effect of PTX formulations assessed using a co-culture model of
MDA-MB-231 cells and hydroxyapatite particles in three-dimensional collagen gel. *P < 0.05, **P < 0.01, ***P < 0.001 compared with PTX group. (n = 3,
mean SD).
biosecurity.
these cells. Interestingly, it was noted that the formulations only
slightly affected the survival of MCF-10A cells (Fig. 3C), which further
suggested the specific effects of the PTX-loaded liposomes on tumors.
Hence, our liposomal drug delivery systems were safe and non-toxic to
be further used in vivo.
What’s more, we further developed an in vitro bone metastasis
model, which was established by co-incubating of MDA-MB-231 cells
and HAP, to estimate the potential of PTX-Glu6-FA-Lip for treating the
bone metastasis from breast cancer. Bone is comprised of bone matrix
proteins and hydroxyapatite and the collagen gel containing HAP was
used to simulate the natural environment of bone for the growth of
breast cancer cells. As shown in Fig. 3D, it was found that the liposome
PTX-Glu6-FA-Lip could significantly inhibit the growth of MDA-MB-231
cells with the lowest cell viability, which revealed the priority of Glu6-
FA functionalization for treating bone metastasis as compared with
other groups (PTX-Lip, PTX-Glu6-Lip, PTX-FA-Lip, and PTX-Glu6+FA-
Lip).
3.3. HAP binding assay
As shown in Fig. 2F, all the liposomes exhibited some HAP affinity,
whereas the free paclitaxel was at best negligibly bound. Moreover,
compared with PTX, PTX-Lip and PTX-FA-Lip, PTX-Glu6-Lip, PTX-
Glu6+FA-Lip and PTX-Glu6-FA-Lip exhibited a significantly higher HAP
binding efficiency with hydroxyapatite power. These results demon-
strated that Glu6-modified liposome had good bone-targeting ability,
and facilitated the next in vivo experiments.
3.4. Cytotoxicity assay
The cytotoxicity of different liposomes on MDA-MB-231 cells and
MCF10A cells was evaluated using MTT assay. As shown in Fig. 3A, the
cytotoxicity of blank liposomes was also measured, and it was shown
that all the kinds of blank liposomes had no significant cytotoxicity at
different concentrations (0.64−2000 ng/mL), and > 90 % of MDA-MB-
231 cells survived. Furthermore, free PTX showed higher inhibition rate
than PTX-loaded liposomes, because free drugs could be transported
into the cells directly by passive diffusion, without a drug release pro-
cess (Fig. 3B). For comparison, we used a normal breast epithelial cell
line, MCF-10A, to examine the effects of the drugs on the survival of
3.5. Cell cycle distribution
The cell cycle is precisely regulated by various regulators to mediate
cell proliferation and survival. We then explored the difference of cell
cycle between control and the different PTX-loaded liposomes treating
breast cancer cells, respectively. As shown in Fig. 4, our results revealed
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Y. Yang, et al.
ChemistryandPhysicsofLipids228(2020)104882
Fig. 4. The PTX formulations induced the arrest of cell cycle
in breast cancer cells. MDA-MB-231 cells were treated with
control or the different PTX-loaded liposomes, and flow cy-
tometry assays were subsequently performed. The percentages
of cells in G1, S, and G2/M phases between control and the
drug treatment groups. (n = 3, mean SD).
that all the PTX liposomes could decrease the percentage of MDA-MB-
231cells in the G1 phases. Compared to the untreated MDA-MB-231
cells, PTX-loaded liposomes treatment cells caused an obvious accu-
mulation of cells at G2/M phase in different extent. In conclusion, the
drug treatment resulted in cell cycle arrest and blocked the proliferation
of breast cancer cells.
effect on the migration of breast cancer cells. As shown in Fig. 5A, the
uncoated liposome (PTX-Lip) had limit effect in antimigration, while
the singly-modified liposomes (PTX-Glu6-Lip, PTX-FA-Lip) could mod-
estly prevent the migration of the MDA-MB-231 cells. Importantly,
following by introducing the dual-targeting ligand (Glu6-FA or
Glu6+FA), the open wounds remained widest, especially the PTX-Glu6-
FA-Lip group. The remained percentage of wound closure of PTX-Glu6-
FA-Lip was 2.48-time, 1.46-time, 2.38-time, 1.40-time compared with
the PTX-Lip, PTX-FA-Lip, PTX-Glu6-Lip, and PTX-Glu6+FA-Lip, re-
spectively, which due to the dual-targeting Glu6-FA as confirmed in
3.6. Wound healing
We further performed wound healing assays to detect its possible
Fig. 5. (A) The antimigration of PTX liposomal formulations on MDA-MB-231 cells. (B) Relative width of open wound after incubation for 12 h. *P < 0.05,
**P < 0.01, and ***P < 0.001. (n = 3, mean SD).
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Y. Yang, et al.
ChemistryandPhysicsofLipids228(2020)104882
Fig. 6. The drug treatment affects microtubule stability of breast cancer cells. (A) Representative immunofluorescence images of MDA-MB-231 cells treated with
control or the dual-targeting liposomes (PTX-Glu6+FA-Lip and PTX-Glu6-FA-Lip) for 48 h followed by incubation on ice for the indicated minutes to depolymerize
microtubules. (B) Experiments were performed as in panel A, and the microtubule intensity was quantified. **P < 0.01, ***P < 0.001 compared with control group.
(n = 3, mean SD).
cytotoxicity assay. Therefore, our in vitro data have witnessed the ob-
vious antimigration effects of PTX-Glu6-FA-Lip on MDA-MB-231 cells.
effects of the two dual-targeting liposomes in microtubule stability.
Interestingly, the treatment of liposome PTX-Glu6-FA-Lip significantly
inhibited microtubule depolymerization on ice compared to the other
group (PTX-Glu6+FA-Lip) (Fig. 6), which suggested that Glu6-FA-
modified liposome could obviously affect cell migration and cell cycle
via the regulation of microtubule stability.
3.7. Fluorescence microscopy
Given that microtubules play a key role in regulating cell migration
and cell cycle, previous report indicated that paclitaxel could partici-
pate in the stabilization of microtubule. We therefore investigated the
9

Y. Yang, et al.
ChemistryandPhysicsofLipids228(2020)104882
Fig. 7. (A) Plasma concentrations of paclitaxel in normal SD rats of different PTX formulations. (B–H) PTX concentration 0.5 h, 1 h, 2 h, 4 h and 8 h after injection of
free PTX, PTX-Lip, PTX-Glu6-Lip, PTX-FA-Lip, PTX-Glu6+FA-Lip and PTX-Glu6-FA-Lip in the heart (B), liver (C), spleen (D), lung (E), kidney (F), normal bone (G) and
bone metastatic lesions (H). *P < 0.05, **P < 0.01, ***P < 0.001. (n = 5, mean SD).
3.8. Pharmacokinetic studies
was increased by about 1.66-time for PTX-Glu6-FA-Lip compared to free
PTX. In addition, it was also shown that PTX-Glu6-FA-Lip could extend
the elimination half-life (t1/2) of free PTX from 257 to 486 min, which
might be due to the modification with the ligand Glu6-FA-Chol and the
hydrophilic Glu6-FA outside to sustain the stability in plasma. There-
fore, the targeted liposomes might be temporarily restored in the
plasma and slowly dissociate from the contents in plasma, and this
consequently lead to the superior circulation time of modified lipo-
somes in vivo.
For pharmacokinetic study, the HPLC analytical method was es-
tablished and validated as our previous report. The pharmacokinetic
profiles of paclitaxel from PTX-loaded liposomal formulations and PTX
solution were compared by measuring the concentration of PTX in rat
plasma after administration at 10 mg/kg. The main pharmacokinetic
parameters were analyzed by DAS 2.0. As shown in Fig. 7A and Table 2,
it was found that dual-modified liposomes (PTX-Glu6+FA-Lip and PTX-
Glu6-FA-Lip) and mono-modified liposomes (PTX-FA-Lip and PTX-Glu6-
Lip) showed similar blood concentration-time curves. The pharmaco-
kinetic parameters of PTX from different formulations are summarized
in Table 2. The results showed that the area under the concentration-
time profile (AUC0-t) of paclitaxel in the five types of liposomes was
3.9. Biodistribution and targeting metastatic bone in vivo
The biodistribution study was conducted in tumor-bearing mice to
determine whether there was preferential targeting of the liposome
PTX-Glu6-FA-Lip to bone metastatic lesions in vivo. The paclitaxel was
much higher than that of naked paclitaxel within 24 h. The (AUC0-t
)
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Y. Yang, et al.
ChemistryandPhysicsofLipids228(2020)104882
Table 2
The pharmacokinetic parameters of different PTX formulations. *P < 0.05, **P < 0.01. (n = 5, mean SD).
AUC(0-t) (μg/mL·min)
MRT (min)
Cmax (μg/mL)
t1/2 (min)
PTX
PTX-Lip
PTX-FA-Lip
PTX-Glu6-Lip
PTX-Glu6+FA-Lip
PTX-Glu6-FA-Lip
9583.57 1567.49
339.52 26.87
386.41 53.24*
399.87 84.25*
415.33 93.27**
423.99 77.45**
442.19 87.62**
58.49 5.91
62.37 9.24
59.33 6.52
65.84 8.75
66.42 7.33
61.36 4.16
257.31 48.52
12870.43 2032.38*
15333.62 2431.65**
13548.29 1752.74*
14931.85 1542.16**
15926.33 2431.20**
370.18 83.19*
452.78 138.62**
398.59 112.37**
424.13 99.82**
486.55 142.33**
quantified in each organ. As shown in Fig. 7, it was observed that a
considerable amount of PTX from paclitaxel solution was accumulated
in the heart (Fig. 7B) and kidney (Fig. 7F) at 2 h post-administration.
While, the uptake was reduced by a factor of four (heart) and a factor of
three (kidney) respectively after 8 h. What’s more, it should be worth
noting that there was a relatively lower drug uptake in other organs
(liver, spleen, lung), especially in lower tibia bone (Fig. 7G and H). For
all the liposome formulations, it was shown that both liver and spleen
had significant uptake in these groups (Fig. 7C and D). Liver and spleen
are considered to be the major macrophage organs and thus, the in-
travenously injected liposomes were eliminated rapidly through the
two organs.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.chemphyslip.2020.
104882.
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